The exploration of the role played by RNA binding proteins in the nervous system continues to be an exciting and productive area of research. Neuronal RNA binding proteins function as alternative splicing factors, regulate mRNA stability and protein translation, and can localize mRNA to dendrites; thus these proteins are key regulators of gene regulation in neurons. RNA binding proteins in the nervous system also have important implications for human health: the fragile X syndrome, spinal muscular atrophy, spinocerebellar ataxia 8 and myotonic dystrophy all involve disregulation of RNA-protein interactions in neurons. The major obstacle in the further understanding of how neuronal RNA binding proteins function has been the identification of RNA substrates. The goal of this proposal is to further define the function of the Nova and Hu families of neuron-specific RNA binding proteins, and central to this aim is the identification of those RNA molecules that bind to Nova and Hu in vivo. Here, we describe new methodology we have developed to systematically and comprehensively identify neuronal RNAs bound by the Nova and Hu proteins. Furthermore, specific hypotheses regarding the interaction of Nova and Hu with these RNAs will be biochemically examined in vitro and in vivo.